The Initiation of Meiotic Sex Chromosome Inactivation Sequesters DNA Damage Signaling from Autosomes in Mouse Spermatogenesis.

Meiotic sex chromosome inactivation (MSCI) is an essential event in the mammalian male germline. MSCI is directed by a DNA damage response (DDR) pathway centered on the phosphorylation of histone variant H2AX at serine 139 (termed γH2AX). The failure to initiate MSCI is linked to complete meiotic arrest and elimination of germ cells; however, the mechanisms underlying this arrest and elimination remain unknown. To address this question, we established a new separation-of-function mouse model for H2ax that shows specific and complete defects in MSCI. The genetic change is a point mutation in which another H2AX amino acid residue important in the DDR, tyrosine 142 (Y142), is converted to alanine (H2ax-Y142A). In H2ax-Y142A meiosis, the establishment of DDR signals on the chromosome-wide domain of the sex chromosomes is impaired. The initiation of MSCI is required for stage progression, which enables crossover formation, suggesting that the establishment of MSCI permits the timely progression of male meiosis. Our results suggest that normal meiotic progression requires the removal of ATR-mediated DDR signaling from autosomes. We propose a novel biological function for MSCI: the initiation of MSCI sequesters DDR factors from autosomes to the sex chromosomes at the onset of the pachytene stage, and the subsequent formation of an isolated XY nuclear compartment-the XY body-sequesters DDR factors to permit meiotic progression from the mid-pachytene stage onward. VIDEO ABSTRACT.

Inactivation of the NHEJ Activity of DNA-PKcs Prevents Fanconi Anemia Pre-Leukemic HSC Expansion.

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and high risk of cancer particularly leukemia. Here we show that inactivation of the non-homologous end-joining (NHEJ) activity of DNA-PKcs prevented DNA damage-induced expansion of FA pre-leukemic hematopoietic stem cells (HSCs). Furthermore, we performed serial BM transplantation to demonstrate that the DNA damage-induced expanded FA HSC compartment contained pre-leukemic stem cells that required the NHEJ activity of DNA-PKcs to induce leukemia in the secondary recipients. These results suggest that NHEJ may collaborate with FA deficiency to promote DNA damage-induced expansion of pre-leukemic HSCs.

Fancd2-deficient hematopoietic stem and progenitor cells depend on augmented mitochondrial translation for survival and proliferation.

Members of the Fanconi anemia (FA) protein family are involved in multiple cellular processes including response to DNA damage and oxidative stress. Here we show that a major FA protein, Fancd2, plays a role in mitochondrial biosynthesis through regulation of mitochondrial translation. Fancd2 interacts with Atad3 and Tufm, which are among the most frequently identified components of the mitochondrial nucleoid complex essential for mitochondrion biosynthesis. Deletion of Fancd2 in mouse hematopoietic stem and progenitor cells (HSPCs) leads to increase in mitochondrial number, and enzyme activity of mitochondrion-encoded respiratory complexes. Fancd2 deficiency increases mitochondrial protein synthesis and induces mitonuclear protein imbalance. Furthermore, Fancd2-deficient HSPCs show increased mitochondrial respiration and mitochondrial reactive oxygen species. By using a cell-free assay with mitochondria isolated from WT and Fancd2-KO HSPCs, we demonstrate that the increased mitochondrial protein synthesis observed in Fancd2-KO HSPCs was directly linked to augmented mitochondrial translation. Finally, Fancd2-deficient HSPCs are selectively sensitive to mitochondrial translation inhibition and depend on augmented mitochondrial translation for survival and proliferation. Collectively, these results suggest that Fancd2 restricts mitochondrial activity through regulation of mitochondrial translation, and that augmented mitochondrial translation and mitochondrial respiration may contribute to HSC defect and bone marrow failure in FA.

The non-homologous end-joining activity is required for Fanconi anemia fetal HSC maintenance.

BACKGROUND: Recent studies have shown that deficiency in the Fanconi anemia (FA) DNA repair pathway enhances the error-prone non-homologous end-joining (NHEJ) repair, leading to increased genomic instability, and that genetic or pharmacological inhibition of the NHEJ pathway could rescue the FA phenotype. METHODS: First, we exposed LSK cells from WT and Fanca-/- mice to DNA-PKcs inhibitor NU7026 or Ku70 knockdown to examine whether inhibition of NHEJ sensitizes Fanca-/- HSPCs to PARP inhibitor (PARPi)- or interstrand crosslinking (ICL)-induced cell death and genomic instability. We then generated DNA-PKcs3A/3AFanca-/- mice to investigate the effect of specific inactivation of NHEJ on fetal HSCs. Lastly, we used two p53 mutant models to test whether specific inactivation of the p53 function in apoptosis is sufficient to rescue embryonic lethality and fetal HSC depletion in Fanca-/- DNA-PKcs3A/3A mice. RESULTS: Inhibition of NHEJ sensitizes HSPCs from Fanca-/- mice to PARP inhibition- and ICL-induced cell death and genomic instability and further decreases Fanca-/- HSPC proliferation and hematopoietic repopulation in irradiated transplant recipients. Specific inactivation of NHEJ activity by the knockin DNA-PKcs3A/3A mutation in two FA mouse models, Fanca-/- and Fancc-/-, leads to embryonic lethality. DNA-PKcs3A/3A causes fetal HSC depletion in developing Fanca-/- embryos due to increased HSC apoptosis and cycling. Both p53-/- and a knockin p53515C mutation, which selectively impairs the p53 function in apoptosis, can rescue embryonic lethality and fetal HSC depletion in Fanca-/- DNA-PKcs3A/3A mice. CONCLUSION: These results demonstrate that the NHEJ pathway functions to maintain Fanconi anemia fetal HSCs.

Rational identification of a Cdc42 inhibitor presents a new regimen for long-term hematopoietic stem cell mobilization.

Mobilization of hematopoietic stem cells (HSCs) from bone marrow (BM) to peripheral blood (PB) by cytokine granulocyte colony-stimulating factor (G-CSF) or the chemical antagonist of CXCR4, AMD3100, is important in the treatment of blood diseases. Due to clinical conditions of each application, there is a need for continued improvement of HSC mobilization regimens. Previous studies have shown that genetic ablation of the Rho GTPase Cdc42 in HSCs results in their mobilization without affecting survival. Here we rationally identified a Cdc42 activity-specific inhibitor (CASIN) that can bind to Cdc42 with submicromolar affinity and competitively interfere with guanine nucleotide exchange activity. CASIN inhibits intracellular Cdc42 activity specifically and transiently to induce murine hematopoietic stem/progenitor cell egress from the BM by suppressing actin polymerization, adhesion, and directional migration of stem/progenitor cells, conferring Cdc42 knockout phenotypes. We further show that, although, CASIN administration to mice mobilizes similar number of phenotypic HSCs as AMD3100, it produces HSCs with better long-term reconstitution potential than that by AMD3100. Our work validates a specific small molecule inhibitor for Cdc42, and demonstrates that signaling molecules downstream of cytokines and chemokines, such as Cdc42, constitute a useful target for long-term stem cell mobilization.

A small molecule p53 activator attenuates Fanconi anemia leukemic stem cell proliferation.

Although p53 mutations are common in solid tumors, such mutations are found at a lower frequency in hematologic malignancies. In the genetic disorder Fanconi anemia (FA), p53 has been proposed as an important pathophysiological factor for two important hematologic hallmarks of the disease: bone marrow failure and leukemogenesis. Here we show that low levels of the p53 protein enhance the capacity of leukemic stem cells from FA patients to repopulate immunodeficient mice. Furthermore, boosting p53 protein levels with the use of the small molecule Nutlin-3 reduced leukemia burden in recipient mice. These results demonstrate that the level of p53 protein plays a crucial role in FA leukemogenesis.

CHEK1 coordinates DNA damage signaling and meiotic progression in the male germline of mice.

The continuity of life depends on mechanisms in the germline that ensure the integrity of the genome. The DNA damage response/checkpoint kinases ATM and ATR are essential signaling factors in the germline. However, it remains unknown how a downstream transducer, Checkpoint Kinase 1 (CHEK1 or CHK1), mediates signaling in the male germline. Here, we show that CHEK1 has distinct functions in both the mitotic and meiotic phases of the male germline in mice. In the mitotic phase, CHEK1 is required for the resumption of prospermatogonia proliferation after birth and the maintenance of spermatogonia. In the meiotic phase, we uncovered two functions for CHEK1: one is the stage-specific attenuation of DNA damage signaling on autosomes, and the other is coordination of meiotic stage progression. On autosomes, the loss of CHEK1 delays the removal of DNA damage signaling that manifests as phosphorylation of histone variant H2AX at serine 139 (γH2AX). Importantly, CHEK1 does not have a direct function in meiotic sex chromosome inactivation (MSCI), an essential event in male meiosis, in which ATR is a key regulator. Thus, the functions of ATR and CHEK1 are uncoupled in MSCI, in contrast to their roles in DNA damage signaling in somatic cells. Our study reveals stage-specific functions for CHEK1 that ensure the integrity of the male germline.

Cell-Cycle-Specific Function of p53 in Fanconi Anemia Hematopoietic Stem and Progenitor Cell Proliferation.

Overactive p53 has been proposed as an important pathophysiological factor for bone marrow failure syndromes, including Fanconi anemia (FA). Here, we report a p53-dependent effect on hematopoietic stem and progenitor cell (HSPC) proliferation in mice deficient for the FA gene Fanca. Deletion of p53 in Fanca-/- mice leads to replicative exhaustion of the hematopoietic stem cell (HSC) in transplant recipients. Using Fanca-/- HSCs expressing the separation-of-function mutant p53515C transgene, which selectively impairs the p53 function in apoptosis but keeps its cell-cycle checkpoint activities intact, we show that the p53 cell-cycle function is specifically required for the regulation of Fanca-/- HSC proliferation. Our results demonstrate that p53 plays a compensatory role in preventing FA HSCs from replicative exhaustion and suggest a cautious approach to manipulating p53 signaling as a therapeutic utility in FA.

Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells.

Bone Marrow Hematopoietic Stem Cells (HSCs) require bone marrow microenvironment for their maintenance and proliferation. Culture of Bone Marrow Mesenchymal Stromal Cells (MSCs) provides appropriate environmental signals for HSCs survival in vitro. Here, we provide a detailed protocol that describes culture conditions for MSCs, flow cytometric isolation of HSCs from mouse bone marrow, and perform co-culture of MSCs and HSCs known as Cobblestone area-forming cell (CAFC) assay. Altogether, CAFC assays can be used as a high-throughput in vitro screening model where efforts are made to understand and develop therapies for complex bone marrow diseases. This protocol needs 3 to 4 weeks starting from culturing MSCs, isolating LSK cells (HSCs), and to performing limited dilution CAFC assay.

Fancd2 in vivo interaction network reveals a non-canonical role in mitochondrial function.

Fancd2 is a component of the Fanconi anemia (FA) DNA repair pathway, which is frequently found defective in human cancers. The full repertoire of Fancd2 functions in normal development and tumorigenesis remains to be determined. Here we developed a Flag- and hemagglutinin-tagged Fancd2 knock-in mouse strain that allowed a high throughput mass spectrometry approach to search for Fancd2-binding proteins in different mouse organs. In addition to DNA repair partners, we observed that many Fancd2-interacting proteins are mitochondrion-specific. Fancd2 localizes in the mitochondrion and associates with the nucleoid complex components Atad3 and Tufm. The Atad3-Tufm complex is disrupted in Fancd2-/- mice and those deficient for the FA core component Fanca. Fancd2 mitochondrial localization requires Atad3. Collectively, these findings provide evidence for Fancd2 as a crucial regulator of mitochondrion biosynthesis, and of a molecular link between FA and mitochondrial homeostasis.

In Vivo RNAi Screen Unveils PPARγ as a Regulator of Hematopoietic Stem Cell Homeostasis.

Hematopoietic stem cell (HSC) defects can cause repopulating impairment leading to hematologic diseases. To target HSC deficiency in a disease setting, we exploited the repopulating defect of Fanconi anemia (FA) HSCs to conduct an in vivo short hairpin RNA (shRNA) screen. We exposed Fancd2-/- HSCs to a lentiviral shRNA library targeting 947 genes. We found enrichment of shRNAs targeting genes involved in the PPARγ pathway that has not been linked to HSC homeostasis. PPARγ inhibition by shRNA or chemical compounds significantly improves the repopulating ability of Fancd2-/- HSCs. Conversely, activation of PPARγ in wild-type HSCs impaired hematopoietic repopulation. In mouse HSCs and patient-derived lymphoblasts, PPARγ activation is manifested in upregulating the p53 target p21. PPARγ and co-activators are upregulated in total bone marrow and stem/progenitor cells from FA patients. Collectively, this work illustrates the utility of RNAi technology coupled with HSC transplantation for the discovery of novel genes and pathways involved in stress hematopoiesis.

Elucidation of the Fanconi Anemia Protein Network in Meiosis and Its Function in the Regulation of Histone Modifications.

Precise epigenetic regulation of the sex chromosomes is vital for the male germline. Here, we analyze meiosis in eight mouse models deficient for various DNA damage response (DDR) factors, including Fanconi anemia (FA) proteins. We reveal a network of FA and DDR proteins in which FA core factors FANCA, FANCB, and FANCC are essential for FANCD2 foci formation, whereas BRCA1 (FANCS), MDC1, and RNF8 are required for BRCA2 (FANCD1) and SLX4 (FANCP) accumulation on the sex chromosomes during meiosis. In addition, FA proteins modulate distinct histone marks on the sex chromosomes: FA core proteins and FANCD2 regulate H3K9 methylation, while FANCD2 and RNF8 function together to regulate H3K4 methylation independently of FA core proteins. Our data suggest that RNF8 integrates the FA-BRCA pathway. Taken together, our study reveals distinct functions for FA proteins and illuminates the male sex chromosomes as a model to dissect the function of the FA-BRCA pathway.

The Fanconi anemia pathway controls oncogenic response in hematopoietic stem and progenitor cells by regulating PRMT5-mediated p53 arginine methylation.

The Fanconi anemia (FA) pathway is involved in DNA damage and other cellular stress responses. We have investigated the role of the FA pathway in oncogenic stress response by employing an in vivo stress-response model expressing the Gadd45ß-luciferase transgene. Using two inducible models of oncogenic activation (LSL-K-rasG12D and MycER), we show that hematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA core complex components Fanca or Fancc exhibit aberrant short-lived response to oncogenic insults. Mechanistic studies reveal that FA deficiency in HSPCs impairs oncogenic stress-induced G1 cell-cycle checkpoint, resulting from a compromised K-rasG12D-induced arginine methylation of p53 mediated by the protein arginine methyltransferase 5 (PRMT5). Furthermore, forced expression of PRMT5 in HSPCs from LSL-K-rasG12D/CreER-Fanca-/- mice prolongs oncogenic response and delays leukemia development in recipient mice. Our study defines an arginine methylation-dependent FA-p53 interplay that controls oncogenic stress response.

Hyper-active non-homologous end joining selects for synthetic lethality resistant and pathological Fanconi anemia hematopoietic stem and progenitor cells.

The prominent role of Fanconi anemia (FA) proteins involves homologous recombination (HR) repair. Poly[ADP-ribose] polymerase1 (PARP1) functions in multiple cellular processes including DNA repair and PARP inhibition is an emerging targeted therapy for cancer patients deficient in HR. Here we show that PARP1 activation in hematopoietic stem and progenitor cells (HSPCs) in response to genotoxic or oxidative stress attenuates HSPC exhaustion. Mechanistically, PARP1 controls the balance between HR and non-homologous end joining (NHEJ) in double strand break (DSB) repair by preventing excessive NHEJ. Disruption of the FA core complex skews PARP1 function in DSB repair and led to hyper-active NHEJ in Fanca(-/-) or Fancc(-/-) HSPCs. Re-expression of PARP1 rescues the hyper-active NHEJ phenotype in Brca1(-/-)Parp1(-/-) but less effective in Fanca(-/-)Parp1(-/-) cells. Inhibition of NHEJ prevents myeloid/erythroid pathologies associated with synthetic lethality. Our results suggest that hyper-active NHEJ may select for "synthetic lethality" resistant and pathological HSPCs.

Loss of Fancc Impairs Antibody-Secreting Cell Differentiation in Mice through Deregulating the Wnt Signaling Pathway.

Fanconi anemia (FA) is characterized by a progressive bone marrow failure and an increased incidence of cancer. FA patients have high susceptibility to immune-related complications such as infection and posttransplant graft-versus-host disease. In this study, we investigated the effect of FA deficiency in B cell function using the Fancc mouse model. Fancc(-/-) B cells show a specific defect in IgG2a switch and impaired Ab-secreting cell (ASC) differentiation. Global transcriptome analysis of naive B cells by mRNA sequencing demonstrates that FA deficiency deregulates a network of genes involved in immune function. Significantly, many genes implicated in Wnt signaling were aberrantly expressed in Fancc(-/-) B cells. Consistently, Fancc(-/-) B cells accumulate high levels of ß-catenin under both resting and stimulated conditions, suggesting hyperactive Wnt signaling. Using an in vivo Wnt GFP reporter assay, we verified the upregulation of Wnt signaling as a potential mechanism responsible for the impaired Fancc(-/-) B cell differentiation. Furthermore, we showed that Wnt signaling inhibits ASC differentiation possibly through repression of Blimp1 and that Fancc(-/-) B cells are hypersensitive to Wnt activation during ASC differentiation. Our findings identify Wnt signaling as a physiological regulator of ASC differentiation and establish a role for the Wnt pathway in normal B cell function and FA immune deficiency.

SCO2 Mediates Oxidative Stress-Induced Glycolysis to Oxidative Phosphorylation Switch in Hematopoietic Stem Cells.

Fanconi anemia (FA) is an inherited bone marrow (BM) failure syndrome, presumably resulting from defects in hematopoietic stem cells (HSCs). Normal HSCs depend more on glycolysis than on oxidative phosphorylation (OXPHOS) for energy production. Here, we show that FA HSCs are more sensitive to the respiration inhibitor NaN3 treatment than to glycolytic inhibitor 2-deoxy-d-glucose (2-DG), indicating more dependence on OXPHOS. FA HSCs undergo glycolysis-to-OXPHOS switch in response to oxidative stress through a p53-dependent mechanism. Metabolic stresses induce upregulation of p53 metabolic targets in FA HSCs. Inactivation of p53 in FA HSCs prevents glycolysis-to-OXPHOS switch. Furthermore, p53-deficient FA HSCs are more sensitive to 2-DG-mediated metabolic stress. Finally, oxidative stress-induced glycolysis-to-OXPHOS switch is mediated by synthesis of cytochrome c oxidase 2 (SCO2). These findings demonstrate p53-mediated OXPHOS function as a compensatory alteration in FA HSCs to ensure a functional but mildly impaired energy metabolism and suggest a cautious approach to manipulating p53 signaling in FA.

Fancb deficiency impairs hematopoietic stem cell function.

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, variable congenital malformations and a predisposition to malignancies. FANCB (also known as FAAP95), is the only X-linked FA gene discovered thus far. In the present study, we investigated hematopoiesis in adult Fancb deficient (Fancb(-/y)) mice and found that Fancb(-/y) mice have decreased hematopoietic stem cell (HSC) quiescence accompanied by reduced progenitor activity in vitro and reduced repopulating capacity in vivo. Like other FA mouse models previously reported, the hematopoietic system of Fancb(-/y) mice is hypersensitive to DNA cross-linking agent mitomycin C (MMC), which induces bone marrow failure in Fancb(-/y) mice. Furthermore, Fancb(-/y) BM exhibits slower recovery kinetics and less tolerance to myelotoxic stress induced by 5-fluorouracil than wild-type littermates. RNA-seq analysis reveals altered expression of genes involved in HSC function and cell cycle regulation in Fancb(-/y) HSC and progenitor cells. Thus, this Fancb(-/y) mouse model provides a novel approach for studying the critical role of the FA pathway not only in germ cell development but also in the maintenance of HSC function.